CB HSPCs expressing BRAF^V600Eengraft MISTRG mice and form clusters with an inflammatory signature in the BM. (A) CB HSPCs were transduced with control or BRAF^V600E virus, sorted according to GFP expression and to RNA sequencing and immunostaining with a specific anti-BRAF^V600E antibody. (B) Western blot analysis showing expression of BRAF^V600E in HeLa cells 5 days after transduction. Expression of BRAF^V600E, phosphorylated Erk (pErk), and total expression of Erk (tErk) were evaluated using specific antibodies. (C) Transduction efficiency of CB HSPCs with control or BRAF^V600E constructs determined by GFP expression level using flow cytometry at day 4 after transduction. (D) Schematic presentation of in vivo experimental procedure. (E) Representative BM FACS analysis of mice 5 weeks after transplantation with control-GFP or BRAF^V600E-GFP transduced human CB HSPCs. FACS plots show frequency of hCD45⁺ (human) cells vs mCD45⁺ (mouse) cells (left panels) and percentage of GFP⁺ cells within hCD45⁺ cells (middle panels). Right panels represent expression of hCD19⁺ and hCD33⁺ cells among GFP⁺ cells. (F) Frequency of engrafted human hCD45⁺ cells, percentage of GFP⁺ cells among hCD45⁺ cells and percentage of hCD19⁺, hCD33⁺ among GFP⁺ cells in BM of adult MISTRG mice. Bars indicate mean values; n = 10 Ctrl, n = 17 BRAF^V600E. (G) Representative BM images of MISTRG mice transplanted with control or BRAF^V600E-transduced human CB HSPCs and analyzed 5 weeks later. Three-dimensional images of entire longitudinal femoral BM slices of mice transplanted with control (a-g) and BRAF^V600E (h-o) CB HSPCs. Low-magnification panoramic views of marrow cavities (a,h) and enlarged, high-resolution images of metaphysis and diaphysis (b-g,i-o). Femoral slices were immuno-stained for collagen IV (turquoise) and endoglin (red). Green color represents GFP, indicating transduced human CB HSPC-derived cells. The distinct morphology of engrafted GFP⁺ cells in mice transplanted with control (f-g) or BRAF^V600E (n-o) is shown (GFP = green, DAPI = white). Imaging depth for Figure 1G: a-e and h-m, 36 µm; f-g and n-o, 4 µm.